小鼠Mac-2结合蛋白酶联免疫吸附测定法的建立及其在小鼠慢性肝病模型中的应用。

Establishment of mouse Mac-2 binding protein enzyme-linked immunosorbent assay and its application for mouse chronic liver disease models.

作者信息

Iwata Ayumi, Kamada Yoshihiro, Ebisutani Yusuke, Yamamoto Akiko, Ueda Yui, Arai Hitomi, Fujii Hironobu, Takamatsu Shinji, Maruyama Nobuhiro, Maeda Masahiro, Takehara Tetsuo, Miyoshi Eiji

机构信息

Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.

Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Japan.

出版信息

Hepatol Res. 2017 Aug;47(9):902-909. doi: 10.1111/hepr.12819. Epub 2016 Oct 13.

DOI:10.1111/hepr.12819

PMID:27615366

Abstract

AIM

We identified Mac-2 (galectin-3) binding protein (Mac-2bp) as a novel diagnostic and liver fibrosis predicting biomarker for nonalcoholic steatohepatitis in humans. In mouse models, there are no serum biomarkers predicting liver disease severity. In this study, we developed a mouse Mac-2bp enzyme-linked immunosorbent assay (ELISA) system and determined its efficacy for predicting the severity of liver disease in mouse models, especially in non-alcoholic fatty liver disease (NAFLD) models.

METHODS

We established several rat monoclonal antibodies against mouse Mac-2bp, selected two clones for the ELISA, and checked the accuracy and reproducibility of the ELISA, especially for NAFLD models and liver fibrosis models. We also investigated the relationships between serum levels and hepatic gene expression of Mac-2bp in mouse models.

RESULTS

Our ELISA system had high accuracy and reproducibility (R = 0.999). The intra-assay and inter-assay results for the coefficient of variation were 2.0-3.7% and 1.7-6.9%, respectively. The levels of bilirubin, hemoglobin, and chyle did not affect the Mac-2bp serum levels detected by our ELISA kit. In the mouse models, serum Mac-2bp levels increased with liver disease progression (F0/F1/F2/F3, 239.1 ± 36.7 / 259.1 ± 43.0 / 457.5 ± 162.0 / 643.7 ± 116.0 ng/mL; P < 0.0001), and were significantly correlated with hepatic gene expression of Mac-2bp (R = 0.42, P < 0.0001).

CONCLUSION

Our mouse Mac-2bp ELISA system effectively predicts severity of NAFLD and liver fibrosis in mouse models.

摘要

目的

我们将Mac-2（半乳糖凝集素-3）结合蛋白（Mac-2bp）鉴定为人类非酒精性脂肪性肝炎一种新型的诊断和预测肝纤维化的生物标志物。在小鼠模型中，尚无预测肝病严重程度的血清生物标志物。在本研究中，我们开发了一种小鼠Mac-2bp酶联免疫吸附测定（ELISA）系统，并确定其在预测小鼠模型尤其是非酒精性脂肪性肝病（NAFLD）模型中肝病严重程度方面的效能。

方法

我们制备了几种抗小鼠Mac-2bp的大鼠单克隆抗体，选择两个克隆用于ELISA，并检测了ELISA的准确性和可重复性，尤其是针对NAFLD模型和肝纤维化模型。我们还研究了小鼠模型中Mac-2bp血清水平与肝脏基因表达之间的关系。

结果

我们的ELISA系统具有高准确性和可重复性（R = 0.999）。批内和批间变异系数结果分别为2.0 - 3.7%和1.7 - 6.9%。胆红素、血红蛋白和乳糜水平不影响我们的ELISA试剂盒检测的Mac-2bp血清水平。在小鼠模型中，血清Mac-2bp水平随肝病进展而升高（F0/F1/F2/F3，239.1±36.7 / 259.1±43.0 / 457.5±162.0 / 643.7±116.0 ng/mL；P < 0.0001），并且与Mac-2bp的肝脏基因表达显著相关（R = 0.42，P < 0.0001）。